As a result of advances in pharmacogenomics, it is now possible to predict the effects or side effects of drugs in individual patients by gene diagnosis based on the relationship between gene polymorphism and drug effects, or between gene polymorphism and side effects. An example is the gene polymorphism of drug metabolizing enzymes. Examples of known drug metabolizing enzymes whose activity is increased or decreased by such polymorphism include cytochrome P4501A2, cytochrome P4502A6, cytochrome P4502C9, cytochrome P4502C19, cytochrome P4502D6, and cytochrome P4502E1. In addition, it has been reported that among a group of enzymes known as conjugation enzymes, such as thiopurine methyltransferase, N-acetyltransferase, UDP-glucuronosyltransferase, or glutathione S-transferase, such gene polymorphism exists, and that the activities of the above enzymes are decreased by such polymorphism (“SNP Idenshi Takei no Senryaku (Strategy of SNP Gene Polymorphism)”, edited by Yusuke Nakamura, Nakayama Shoten, Jun. 5, 2000).
Moreover, by examining the relationship between gene polymorphism and diseases, the pre-diagnosis of several diseases or the determination of prognosis becomes possible. A large number of disease-associated genes discovered as a result of polymorphism analyses have been reported. Examples of such disease-associated genes, which have been reported, include: HLA, a causative gene of ulcerative colitis; TCRα, a causative gene of rheumatoid arthritis; APOE4, a causative gene of Alzheimer's disease; a dopamine D3 receptor, a causative gene of schizophrenia; tryptophan hydroxylase, a causative gene of manic-depressive psychosis; an angiotensin precursor, a causative gene of albuminuria; blood coagulation factor VII, a causative gene of myocardial infarct; and leptin, a causative gene of adiposis (Nature Genetics, 1999, Vol. 22, pp. 139-144).
Examples of methods for detecting gene polymorphism, which have been developed, include: the PCR-RFLP method, involving a combination of the polymerase chain reaction (PCR) method and cleavage with restriction enzymes (Science, 1991, Vol. 252, p. 1643-); the SSCP (single-strand conformation polymorphism) method, based on the principle that single-strand DNA and RNA having different sequences exhibit different electrophoretic mobility in polyacrylamide gels; and the AS-PCR (allele-specific PCR) method, based on the principle that mismatches existing at the 3′-end of an oligonucleotide primer inhibit elongation of the primer.
Since the PCR-RFLP method comprises a treatment with restriction enzymes for 3 to 24 hours in its test process, it is difficult to say that this is a rapid method. The SSCP method is excellent in that when one or several mutations exist in any part of the nucleotide sequence used as a test target, this method is able to detect such existence at high sensitivity. However, since the experimental conditions are strictly controlled to detect a subtle difference in mobility, this is extremely complicated, and furthermore, the position of the mutation cannot be identified by this method. In addition, in order to perform the SSCP method using actual analytes such as blood or tissues, it is necessary to prepare, in advance, a large amount of nucleic acid via cloning or the PCR method. Thus, this method is not suitable for efficiently testing a large number of analytes.
The AS-PCR method is a method that involves a modification of PCR. For this method it is not necessary to prepare in advance a large amount of nucleic acid. This method is based on the fact that an amplified product can be obtained only when primers having no mismatch around the 3′-ends thereof are used. This is a method suitable for efficiently testing a large number of analytes. However, there are cases where such an amplified product can be obtained in ordinary PCR, even when mismatches exist in primers. Thus, the above method has been problematic in terms of stringency.
Also, it has been reported that when the above AS-PCR method is modified, and when a primer having a nucleoside with a base that is not complementary to the target gene at the second position from the 3′-end is prepared, and the polymorphic portion to be detected is set at the 3′-end thereof, when compared with a primer having a nucleoside with a base complementary to the target gene at the second position from the 3′-end, detection of the polymorphic portion existing at the 3′-end is improved (Bioorganic & Medical Chemistry, 2003, Vol. 11, pp. 2211-2226). However, even when using this method, there are cases in which an amplified product can be obtained even if mismatch exists at the 3′-end of the primer. Accordingly, development of a method for detecting gene polymorphism at higher detection sensitivity is desired.
A 2′-O,4′-C-ethylene nucleotide (hereinafter referred to as an “ENA nucleotide” at times) is a non-natural nucleotide. An oligonucleotide into which such an ENA nucleotide has been introduced has high binding ability to complementary strand RNA (Japanese Patent No. 3420984 (Japanese Patent Laid-Open No. 12-297097) and Bioorganic & Medical Chemistry, 2003, Vol. 11, pp. 2211-2226). In addition, the ENA nucleotides are characterized in that they have a higher resistance to nuclease than LNA nucleotides (2′-O,4′-C-methylene nucleotide (Japanese Patent Laid-Open No. 10-304889), which are formed by crosslinking, with a methylene chain, an oxygen atom at the 2′ position and a carbon atom at the 4′ position of a sugar portion (Bioorganic & Medicinal Chemistry Letters, 2002, Vol. 12, pp. 73-76). However, it was not known if the use of ENA nucleotides in a primer would improve the sensitivity of AS-PCR.
As a result of studies directed towards solving the aforementioned problems with polymorphism detection methods, the present inventors have found that when an oligonucleotide used as a PCR primer has a polymorphic portion at the 3′-end thereof and the third position from the 3′-end is modified with ENA, the amount of an amplified product generated due to mismatches is decreased, and gene polymorphism can be detected with high precision. The inventors have further provided a kit for use in the above detection method.
Moreover, the present inventors have also found that when an oligonucleotide used as a PCR primer has a polymorphic portion at the 3′-end, a nucleotide having a base that is not complementary to a gene to be detected as the second nucleotide from the 3′-end, and the third position from the 3′-end is modified with ENA, the amount of an amplified product generated due to mismatches is decreased, and gene polymorphism can be detected with high precision. The inventors have further provided a kit for use in the above detection method, thereby completing the present invention.